酚类物质暴露对男性生殖危害的流行病学与代谢组学研究

发布时间：2018-01-19 11:30

本文关键词：双酚A 氯酚二苯甲酮-3 烷基酚尿液超高效液相色谱串联质谱双酚A 4-t-辛基酚 4-n-辛基酚 4-n-壬基酚男性不育精液参数 4-t-辛基酚 Y染色体单倍群男性不育易感性代谢组学睾丸尿液双酚A 生殖毒性　出处：《南京医科大学》2013年博士论文　论文类型：学位论文

【摘要】：酚类物质是一类典型的环境内分泌干扰物,人群暴露广泛。以往的动物和体外实验发现酚类物质暴露与男性生殖损害有关,然而这些研究缺乏人群流行病学证据支持且生殖毒性机制尚未阐明。男性不育是环境因素和基因因素共同作用的结果,故而环境-基因交互作用是目前研究化学物毒性易感性的研究热点。另外,代谢组学作为一门新兴的组学技术,在生殖毒理学研究中具有极大的潜力与价值。故而本研究关注酚类物质暴露与男性生殖,采用环境与遗传并进、人群加动物的整体研究策略。我们先建立了基于超高效液相色谱串联质谱检测人尿液中酚类物质的方法;并借助该方法检测了中国普通人群尿液中酚类物质暴露水平,探讨了酚类物质暴露与男性生殖的流行病学关联;并进一步分析了代表性酚类物质4-t-辛基酚的生殖毒性在不同Y染色体单倍群背景下易感性的差异;最后采用代表性酚类物质双酚A的大鼠染毒模型,开展了多分析平台联合的睾丸和尿液的代谢组学研究,发现了双酚A暴露的生物标志并揭示了双酚A暴露对雄性生殖损伤的毒性机制。这些研究为酚类物质的潜在男性生殖毒性提供了人群流行病学证据和全新的认识,为酚类物质的男性生殖危害评价提供了基础资料。第一部分酚类物质暴露与男性生殖异常的流行病学研究第一节尿液中酚类物质检测方法的开发前言酚类物质的暴露是否危害人类健康是重要的公共卫生问题。然而,同时检测多种酚类物质的内暴露水平的方法还很缺乏。本研究基于超高效液相色谱串联质谱技术首次开发了检测人尿液中九种酚类物质的方法,包括双酚A,2,3,4-三氯酚、2,4,5-三氯酚、二苯甲酮-3、五氯酚、三氯生、4-t-辛基酚、4-n-辛基酚和4-n-壬基酚。方法1.采用β-葡糖苷酸-芳基硫酸酯酶酶解尿液;2.采用Envi-18固相萃取小柱提取和预浓缩目标酚类物质;3.采用超高效液相色谱串联质谱分离目标酚类物质并采用多反应监测模式进行酚类物质定量检测。结果1.方法的灵敏度:检测限0.02-0.9μg/l;2.方法的稳定性:日内精密度2-41%,日间精密度3-38%;3.方法的回收率:78-138%;4.方法线性范围:0.03-50μg/l;5.基于此方法检测了325名来自中国普通人群的志愿者尿液中酚类物质的含量,发现中国普通人群有较为普遍的酚类物质暴露。结论本研究首次开发了基于超高效液相色谱串联质谱技术的高通量检测尿液中酚类物质的灵敏、准确、稳定的方法,为酚类物质内暴露水平检测和酚类物质暴露与疾病关联的流行病学研究提供了可行有效的检测方法。本部分研究还首次报道了中国人群酚类物质内暴露水平的基线资料。第二节酚类物质暴露与男性不育及精液参数异常的相关性研究前言酚类物质具有内分泌干扰效应,体外和动物实验广泛报道其暴露与男性生殖损伤有关,然而人群中酚类物质暴露是否与男性不育有关依然缺乏流行病学证据。本研究采用病例对照的设计研究酚类物质暴露与男性不育及精液参数异常的相关性。方法1.采用已开发的基于超高效液相色谱串联质谱技术的方法分析中国普通人群877例男性不育及713例正常生育对照尿液中酚类物质的浓度;2.对上述人群的精液参数进行分析;3.用多元Logistic回归和趋势性检验分析酚类物质暴露与男性不育及精液参数异常的相关性。结果1.中国普通人群的男性有较为普遍的酚类物质的暴露,暴露水平从高到低依次是三氯生、五氯酚、二苯甲酮-3、4-t-辛基酚、4-n-壬基酚和4-n-辛基酚。2.4-t-辛基酚、4-n-辛基酚和4-n-壬基酚暴露与男性不育有关,趋势性检验p值分别为0.0001,0.014和0.001。3.双酚A暴露、4-t-辛基酚和4-n-壬基酚暴露与异常精液参数有关。4.烷基酚联合暴露(4-t-辛基酚、4-n-辛基酚和4-n-壬基酚)与男性不育及异常精液参数有关。结论本研究首次基于大样本人群探讨了酚类物质暴露与男性不育及精液参数异常的相关性,为酚类物质的男性生殖毒性提供了流行病学证据。第三节代表性酚类物质男性生殖毒性的遗传易感性研究前言男性不育是环境因素和遗传因素共同作用的结果。4-t-辛基酚暴露与男性不育及异常精液参数在中国普通人群具有较强的关联,故而进一步探讨不同遗传背景下的人群对4-t-辛基酚的生殖毒性的易感性差异对进一步深化对4-t-辛基酚的男性生殖毒性认识具有重要的意义。Y染色体单倍群与男性生殖密切相关,在遗传背景研究中非常重要。本研究采用病例对照研究设计探讨Y染色体单倍群对代表性酚类物质(4-t-辛基酚)生殖毒性的易感性的影响。方法1.采用已开发的基于超高效液相色谱串联质谱技术的方法分析了774例男性不育及520例正常生育对照尿液中4-t-辛基酚的浓度;2.在上述人群中采用SNa Pshot方法分类了10个Y染色体单倍群;3.用似然比检验分析Y染色体单倍群的对4-t-辛基酚生殖毒性易感性的影响,计算假阳性率来控制多重检验假设带来的假阳性错误率。结果1.4-t-辛基酚的暴露在本研究各Y染色体单倍群中均提高了男性不育的风险。2.Y染色体单倍群O3*人群相比非本群男性对4-t-辛基酚的潜在生殖毒性不易感(pinter=1.1e-3)。结论本研究首次分析了十种Y染色体单倍群下4-t-辛基酚生殖毒性易感性的分布情况,说明了中国男性普遍对4-t-辛基酚的生殖毒性易感,揭示了O3*单倍群一定程度上降低了4-t-辛基酚致男性不育的风险。本研究强调了以后需要更为广泛地开展Y染色体单倍群相关的功能研究和Y染色体单倍群与化学物毒性的易感性研究。第二部分代谢组学在代表性酚类物质致雄性生殖毒性机制研究中的应用前言流行病学研究发现双酚A暴露与精子发生异常有关,而双酚A的生殖毒性机制尚未完全阐明。代谢组学在生殖毒理学研究中具有应用潜力,尚无有关代谢组学应用于双酚A生殖毒性研究的报道。本研究选择双酚A为代表性酚类物质,采用大鼠染毒双酚A模型,分析睾丸和尿液代谢谱变化,探索双酚A的生殖毒性机制。方法1.将18只大鼠随机分为1个溶剂对照组和2个双酚A染毒组(0.0005,50mg/kg/d),采用灌胃法连续染毒8周;2.睾丸和尿液采用液相色谱串联四级杆飞行时间质谱进行非靶标代谢组学分析,采用精确分子量、同位素分布、数据库比对、标准品比对等多种方法鉴定生物标志;3.根据睾丸代谢谱提示的信息,用分光光度法试剂盒测定睾丸超氧物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶活性;4.尿液采用毛细管电泳串联飞行时间质谱进行高通量代谢谱分析,采用标准品库定性内源性化学物;5.肝脏蛋氨酸腺苷转移酶Iα(Mat1a)和蛋氨酸腺苷转移酶IIα(Mat2a)的m RNA和蛋白水平分别采用RT-PCR和Western blot检测;6.计算假阳性率来控制多重检验假设带来的假阳性错误率,用KEGG数据库对结果进行生物信息学分析。结果1.双酚A染毒组尿液中葡萄糖醛酸苷结合型双酚A显著上升;2.睾丸代谢谱:采用多种方法鉴定出了双酚A暴露大鼠睾丸的潜在生物标志-亚油酸和花生四烯酸,双酚A暴露对大鼠睾丸不饱和脂肪酸的代谢产生了影响使得花生四烯酸上升,亚油酸下降,花生四烯酸/亚油酸比值上升,大鼠超氧物歧化酶活性在双酚A染毒后发生显著下降,谷胱甘肽过氧化物酶和过氧化氢酶活性出现了下降趋势;3.尿液代谢谱:双酚A导致甲基化氨基酸、甲基化嘌呤上升,尿素循环增强,核酸降解增强,水溶性维生素排出增多,胆碱相关代谢增强,肝脏编码胆碱代谢通路关键酶的基因mat1a和mat2a的m RNA和蛋白水平均显著上升。结论本研究发现了代表性酚类物质双酚A暴露的睾丸生物标志,双酚A影响了睾丸的脂肪酸代谢,降低了睾丸的抗氧化能力;通过增强胆碱代谢相关关键代谢酶的基因表达,增强胆碱代谢引起了体内高甲基化环境导致异常的蛋白降解和DNA损伤效应从而可能引起生殖毒性。本研究首次从代谢组学角度为双酚A的生殖毒性提供了新的视角,并说明了代谢组学在生殖毒理学研究领域的潜在意义和应用价值。
[Abstract]:Phenol is a kind of typical environmental endocrine disruptors, population exposure widely. The previous animal and in vitro experiments found that the damage of phenolics exposure and male reproductive related, however these studies lack of epidemiological evidence supports and reproductive toxicity mechanism has not been elucidated. Male infertility is the result of interactions between environmental factors and genetic factors, and environment gene interaction is a hotspot of the research on susceptibility to toxicity of chemicals. In addition, metabolomics is an emerging technology, has great potential and value in the study of reproductive toxicology. Therefore this research focuses on the phenolics exposure and male reproductive, with genetic and environmental research, the overall strategy and the crowd animal. We first established a method of ultra performance liquid chromatography tandem mass spectrometry in human urine based on phenolic compounds; and using this method to detect Chinese general population of phenolic substances in the urine level of exposure and discusses the phenol exposure epidemiological association with male reproduction; and further analyzes the differences in the reproductive toxicity of representative phenols 4-t- octylphenol in different Y chromosome haplogroup background susceptibility; the poisoning rats model representative of phenolic compounds of bisphenol A the group carried out metabolic analysis platform with testicular and urine studies, found biomarkers of exposure to bisphenol A and bisphenol A exposure reveals the toxic mechanism of injury to male reproductive potential. These studies for male reproductive toxicity of phenolic substances to provide epidemiological evidence and new understanding, provide the basic data for male reproductive hazard evaluation of phenolic compounds. The first part phenols exposed examination phenolic compounds and epidemiological studies of male reproductive abnormalities section in urine The development of exposure measurement methods of phenolic compounds are harmful to human health is an important public health problem. However, the method of simultaneous detection of multiple exposure levels of phenolic compounds in is still lacking. This research method based on ultra performance liquid chromatography tandem mass spectrometry was developed for the first time to detect the urine of nine kinds of phenolic substances. Including bisphenol A, 2,3,4- three 2,4,5- three two chlorophenols, chlorophenols, benzophenone -3, PCP, three triclosan, 4-t- 4-n- octylphenol, nonylphenol and Octylphenol 4-n-. Methods 1. the beta glucuronide - arylsulfatase enzyme urine; 2. using Envi-18 solid phase extraction column extraction and preconcentration of target phenols material; 3. using ultra performance liquid chromatography tandem mass spectrometry was used to separate target phenols and phenolic compounds detected using multiple reaction monitoring mode. The sensitivity results of 1. methods: the detection limit of 0.02-0.9 g/l; the stability of 2. methods: Date Precision in 2-41%, precision 3-38%; 3. methods of recovery rate: 78-138%; 4. linear range: 0.03-50 g/l 5.; based on this method to detect the content of phenolic compounds in 325 from the general population China volunteers in the urine, ordinary people have found China phenols more general conclusions of this exposure. The first study developed a high-throughput tandem mass spectrometry UPLC detection of phenols in urine based on sensitive, accurate, and stable method for phenols in exposure level detection and phenolics exposure and disease associated epidemics provide a feasible and effective method to detect research. This part of the study it was first reported that the baseline data on exposure levels of phenolic compounds in the Chinese population. The second section phenolics exposure and male infertility and abnormal semen parameters to study the correlation between the phenolic compounds with endocrine disturbance Effect of in vitro and animal experiments reveal the widely reported damage is related to male fertility, however phenols crowd exposure is associated with male infertility is still a lack of epidemiological evidence. This study used a case-control design study on phenolics exposure associated with male infertility and abnormal semen parameters. Methods 1. methods using ultra performance liquid chromatography chromatography tandem mass spectrometry has been developed based on the analysis of China general population of 877 cases of male infertility and 713 cases of normal birth control the concentration of phenols in urine; analysis of semen parameters of 2. of this population; 3. with multivariate Logistic regression and trend test analysis of phenolic substances exposure associated with male infertility and abnormal semen parameters results 1. of the general population. China men exposed phenols widely, the exposure level from high to low are three triclosan, PCP, two Benzophenone -3,4-t- octylphenol, nonylphenol and Octylphenol 4-n- 4-n-.2.4-t- octyl phenol, octyl phenol 4-n- and 4-n- nonylphenol exposure associated with male sterility, trend test p values were 0.0001,0.014 and 0.001.3. exposure to bisphenol A, 4-t- octylphenol and 4-n- nonylphenol exposure and abnormal semen parameters related.4. alkylphenol combined exposure (4-t- octylphenol 4-n- 4-n-, octylphenol and nonylphenol) associated with male infertility and abnormal semen parameters. Conclusion this is the first population-based study based on phenol exposure associated with male infertility and abnormal semen parameters, to provide epidemiological evidence for male reproductive toxicity of phenolic compounds. The third section represents the genetic susceptibility of the male sterility phenolics in male reproductive toxicity is the interaction of environmental and genetic factors results.4-t- octylphenol exposure and male infertility and abnormal semen parameters Has a strong correlation in the general population China, therefore to further explore the different genetic background of the population differences in susceptibility to reproductive toxicity in 4-t- of octyphenol further deepening of male reproductive toxicity of 4-t- octylphenols understanding of the significance of.Y chromosome haplogroup and male reproductive is closely related to the genetic background is very important in the research. The a case-control study design to investigate the Y chromosome haplogroups of representative phenolic compounds in this study (4-t- octylphenol) affect susceptibility to reproductive toxicity. Methods 1. methods using ultra performance liquid chromatography tandem mass spectrometry has been developed based on the analysis of 774 cases of male infertility and 520 cases of normal birth control 4-t- concentration of Octyl phenol in urine 2. in the crowd; using SNa Pshot method for the classification of 10 Y chromosome haplogroup; 3. with likelihood ratio test analysis of Y chromosome haplogroup of 4-t- octyl Effect of phenol on reproductive toxicity susceptibility, calculate the false positive rate to control the false positive error rate caused by multiple hypothesis testing. The results of octylphenol exposure of 1.4-t- compared to the non potential reproductive toxicity of this group of male 4-t- octylphenol in this study are not susceptible to the Y chromosome haplogroups were increased the risk of male infertility.2.Y chromosome group O3* group (pinter=1.1e-3). Conclusion this is the first study to analyze ten kinds of distribution of Y chromosome haplogroup 4-t- octylphenols reproductive toxicity susceptibility, the China men generally on the reproductive toxicity of octylphenol 4-t- susceptible, haplogroup O3* reveals to some extent reduce the risk of male infertility caused by 4-t- octylphenol. This study highlights the need for more extensive future study on susceptibility of Y chromosome haplogroup related function research and the Y chromosome haplogroup and the toxicity of chemicals. In the second part, metabolism Study on representative phenols induced by the application of epidemiological study on Mechanism of male reproductive toxicity study found in exposure to bisphenol A is associated with abnormal sperm, and the reproductive toxicity mechanism of bisphenol A has not been fully elucidated. Metabonomics has potential application in the study of the reproductive toxicology, there is no relevant application of metabonomics to study the reproductive toxicity of bisphenol A reported. This study is representative of bisphenol A phenol, bisphenol A by rat model, analysis of the changes of testis and urine metabolic profiles, reproductive toxicity mechanism of bisphenol A. Methods 1. 18 rats were randomly divided into 1 control groups and 2 bisphenol A exposure group (0.0005,50mg/kg/d), by gavage method after 8 weeks; 2. testis and urine series four stages of rod using time-of-flight mass spectrometry non target metabolomic analysis of liquid chromatography, the accurate molecular weight, isotope distribution, ratio of database The sign of a variety of methods, comparison and identification of biological standards; 3. according to testicular metabolism spectrum prompt information, spectrophotometric determination kit testicular superoxide dismutase, glutathione peroxidase and catalase activity; 4. urine by capillary electrophoresis for high-throughput metabolic profiling of tandem time-of-flight mass spectrometry, using the standard library qualitative endogenous chemicals; 5. liver methionine adenosyltransferase I alpha (Mat1a) and methionine adenosyltransferase II alpha (Mat2a) m RNA and protein level respectively by RT-PCR and Western blot detection; 6. calculation of false positive rate to control the false positive error rate caused by multiple hypothesis testing, bioinformatics analysis of results with the KEGG database. The glucuronide 1. bisphenol A exposure group in urine with bisphenol A increased significantly; 2. testicular metabolic spectrum: using a variety of methods to identify bisphenol A exposure rats The potential biomarkers in testis of linoleic acid and four arachidonic acid, exposure to bisphenol A on rat testis unsaturated fatty acid metabolism affected the four arachidonic acid increased, linoleic acid decreased four arachidonic acid / linoleic acid ratio increased, rat superoxide dismutase activity in bisphenol A after exposure to a significant decline, glutathione peroxidase and catalase activity decline; 3. urine metabolic profiles: bisphenol A causes methylated amino acids, methylation of purine nucleic acid increased, the urea cycle increased, enhanced degradation of water soluble vitamin excretion increased, choline metabolism enhanced liver choline metabolism pathway key enzyme encoding genes mat1a and mat2a m RNA and protein levels were significantly increased. Conclusion this study found testis biomarkers representative phenols exposure to bisphenol A, bisphenol A of fatty acid metabolism in testis, reduced testosterone Pill antioxidant capacity; by enhancing the expression of key metabolic enzymes choline metabolism genes, increased choline metabolism caused in high methylation environment lead to abnormal protein degradation and DNA damage which may cause reproductive toxicity. This is the first study from the aspect of metabonomics provides a new perspective for the reproductive toxicity of bisphenol A, and the metabonomics in reproductive toxicology research potential significance and application value.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114

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